Antagonists of chaperonin 10

ABSTRACT

An antagonist to, or an antibody (Ab) raised against, cpn10 or a recombinant cpn10 with the sequence: GSAGQAFRKFLPLFDRVLVERSAAETVTKGGIMLPEKSQGKVLQATVEAVGSGSKGKGGEIQPVSVKEGDK VLLPEYGGTKVVLDDKDYFLFRDGDILGKYVD is claimed. Also, claimed are: (1) an antagonist or Ab raised against a peptide derived from cpn10, or a peptide with the sequence: Ac-AGQAFRKLPL(C) AGQAFRKFLPLA2 A1AGQAFRKFLPL Ac-A1AGQAFRKFLPL (A1)EKSQGKVLQATA2 A1EKSQGKVLQAT where A1 and A2 are amino acid sequences that may be added to one or both ends of the peptides, and where the peptides may have a single amino acid deletion, addition or substitution; (2) suppressing cellular growth or enhancing immunological activity by admin. of a cpn10 antagonist or anti-cpn10 Ab to a subject; and (3) an assay for measuring anti-cpn10 Ab in a sample by: (a) reacting purified cpn10 with the sample (b) determining the amt. of Ab in the sample by determining the binding between the Ab and cpn10. 
     USE--The cpn 10 antagonist or Ab can be used to terminate pregnancy, suppressing tumour cell growth or enhancing the immune system.

This application is a C-I-P of PCTAU94/00742 filed Apr. 30, 1994 (nowABN).

FIELD OF THE INVENTION

This invention relates to antagonists to chaperonin 10 otherwise knownas cpn10.

PRIOR ART

Chaperonins belong to a wider class of molecular chaperones, moleculesinvolved in post-translational folding, targeting and assembly of otherproteins, but which do not themselves form part of the final assembledstructure as discussed by Ellis et al., 1991, Annu. Rev. Biochem. 60321-347. Most molecular chaperones are "heat shock" or "stress" proteins(hsp); i.e. their production is induced or increased by a variety ofcellular insults (such as metabolic disruption, oxygen radicals,inflammation, infection and transformation), heat being only one of thebetter studies stresses as reviewed by Lindquist et al., 1988, Annu.Rev. Genet. 22 631-677. As well as these quantitative changes inspecific protein levels, stress can induce the movement ofconstitutively produced stress proteins to different cellularcompartments as referred to in the Lindquist reference mentioned above.The heat shock response is one of the most highly conserved geneticsystem known and the various heat shock protein families are among themost evolutionarily stable proteins in existence. As well as enablingcells to cope under adverse conditions, members of these familiesperform essential functions in normal cells.

There are two types of cpn molecules, cpn60 (monomeric M_(r) ˜60,000)and cpn10 (monomeric M_(r) ˜10,000). Cpn60 has been studied extensively.It has been identified in all bacteria, mitochondria and plastidsexamines, and a cytoplasmic form, TCP-1, has been identified ineukaryotic cells: its presence on the surface of some cells has beenreported, although this has been questioned in the Ellis referencereferred to above and also in van Eden, 1991. Immunol. Reviews 121 5-28.Until very recently, cpn10 had been identified only in bacteria butstructural and functional equivalents have now been found inchloroplasts (Bertsch et al., 1992, Proceedings of the National Academyof Sciences USA 89 8696-8700) and in rat (Hartman et al., 1992,Proceedings of the National Academy of Sciences USA 89 3394-3398) andbovine liver mitochondria (Lubben et al., 1990, Proceedings of theNational Academy of Sciences USA 87 7683-7687).

Cpn60 and cpn10 interact functionally, in the presence of ATP, tomediate protein assembly. Instances of cpn10 acting independently ofcpn60 have not yet been reported but cpn60, apparently acting alone, hasbeen implicated in quite disparate events. For example, it is animmuno-dominant target of both antibody and T-cell responses duringbacterial infections but, because the protein is so highly conserved,self reactivity is generated. Healthy individuals may use thisself-recognition to eliminate transformed and infected autologous cellsbut defects in control of such recognition may lead to autoimmunedisease as discussed by van Eden, 1991, Immunol. Reviews 121 5-28. Notsurprisingly, cpn60 has been associated with conditions such asrheumatoid arthritis. There is thus a growing awareness that molecularchaperones, with their capacity to bind to and alter the conformation ofa wide variety of polypeptides, may occupy key roles in cellularfunctions other than protein biogenesis. Reference may also be made toHartman et al., 1993, Proceedings of the National Academy of SciencesUSA 90 2276-2280 which describes the stabilization of protein moleculesusing cpn10 and cpn60.

It can also be established that for mammalian cpn10's, there is a veryclose sequence homology. Thus, for example, the rat cpn10 molecule(Hartman et al., 1992, Proceedings of the National Academy of SciencesUSA 89 3394-3398) has greater than 99% homology with the structure ofbovine cpn10 reported in EMBL Data Base Directory under MT BTC PN10which was submitted by J. E. Walker, MRC Lab. of Molecular Biology,Hills Road, Cambridge, UK. This has to be contrasted with bacterialcpn10's which have an average degree of homology with rat chaperonin 10of only 35% (Hartman et al., 1992).

EARLY PREGNANCY FACTOR (EPF)

EPF was first described as a pregnancy associated substance (Morton etal., 1976, Proc. R. Soc. B. 193 413-419) and its discovery createdconsiderable interest as it enabled the detection of a potentialpregnancy within 6-24 hours of fertilisation. Initially EPF was assigneda role as an immuno-suppressant by virtue of its ability to releasesuppressor factors from lymphocytes (Rolfe et al., 1988, Clin. exp.Immunol. 73 219-225). These suppressor factors depress the delayed typehypersensitivity reaction in mice and therefore might suppress apossibly maternal immune response against the antigenically alien fetus.More recent studies have shown that production of EPF is not confined topregnancy. It is a product of primary and neoplastic cell proliferationand under these conditions acts as a growth factor (Quinn et al., 1990,Clin. exp. Immunol. 80 100-108; Cancer Immunol. Immunother, 1992, 34265-271). EPF is also a product of platelet activation and it isproposed therefore that it may play a part in wound healing and skinrepair (Cavanagh et al., 1991, Journal Reproduction and Fertility 93,355-365).

To date, the rosette inhibition test remains the only means of detectingEPF in complex biological mixtures (Morton et al., 1976, Proc R Soc B413-419). This assay is dependent on the original finding of Bach andAntoine, 1968, Nature (Lond) 217 658-659 that an immunosuppressiveanti-lymphocyte serum (ALS) can inhibit spontaneous rosette formation invitro between lymphocytes and heterologous red blood cells. Amodification of the assay was introduced to detect EPF after it wasdemonstrated that lymphocytes, preincubated in EPF, give a significantlyhigher rosette inhibition titre (RIT) with an ALS than do lymphocytesfrom the same donor without EPF as described in the 1976 referenceabove. This test has been described in detail in the above 1976reference as well as in Morton et al., 1987, in "In Current Topics inDevelopmental Biology" Vol 23 73-92, Academic Press, San Diego, butbriefly it involves a cascade of events with EPF binding to lymphocytesand sequentially inducing the release of suppressor factors (Rolfe etal., 1988, Clin. exp. Immunol. 73 219-225); (Rolfe et al., 1989,Immunol. Cell Biol. 67 205-208).

In Athanasas-Platsis et al., 1989, Journal Reproduction and Fertility 87495-502 and Athanasas-Platsis et al., 1991, Journal Reproduction andFertility 92 443-451, there is described the production of monoclonaland polyclonal antibodies to EPF and passive immunization of pregnantmice with these antibodies which causes loss of embryonic viability.These studies established that EPF is necessary for the successfulestablishment of pregnancy.

In Quinn et al., 1990, Clin. exp. Immunol. 80 100-108, it is proposedthat EPF is a growth regulated product of cultured tumour andtransformed cells. These cells are also dependent upon EPF for continuedgrowth i.e. EPF acts in an autocrine mode.

It has been established that EPF plays a role in promoting tumour growthsince the growth of tumour cells can be significantly retarded byanti-EPF mAbs. In addition this reference suggests that hybridomasproducing high affinity anti-EPF antibodies may be inherently unstable.

In Quinn et al., 1992, Cancer Immunol. Immunother. 34 265-271, there isalso described the effect of monoclonal antibodies (mAbs) to EPF on thein vivo growth of transplantable murine tumours. The main thrust of thisreference is that neutralisation of EPF retards tumour growth in vivo.

It is clear from the above Quinn et al. 1992 reference that when canceris in the very early stage of growth, neutralisation of EPF by anti-EPFmAb will prevent its development. However, once the cancer becomesestablished, treatment with these mAbs will retard but not entirelydestroy the tumour.

Other references in regard to the role of EPF in tumour growth includeQuinn, 1991, Immunol. Cell Biol. 69 1-6 and Quinn, K. A. in a PhD thesisentitled "Early pregnancy factor: a novel factor involved in cellproliferation" from the University of Queensland in Australia in 1991.

EPF is reviewed in detail by Morton et al., 1992, Early PregnancyFactor, Seminars in Reproductive Endocrinology 10 72-82. The site andregulation of EPF production is described, followed by the purificationof EPF from platelets and the relationship of the purified product toEPF derived from other sources. This review also discusses certainaspects of the bioassay for EPF (i.e. the rosette inhibition test)including monitoring purification procedures and investigating sourcesof production. The biological activity of EPF is discussed and possibleclinical applications of EPF and its antagonists are described.

Morton et al., 1992, Reprod. Fertil Dev. 4 411-422 reviews previouspublications describing the immuno suppressive and growth factorproperties of EPF. The role of EPF in maintaining the pre-embryo is alsodiscussed in this reference.

Both of the abovementioned references, which are essentially reviewarticles, describe the preparation of purified EPF for blood plateletswhich included the initial sequential steps of heat extraction of theplatelets, cation exchange chromatography on SP-Sephadex C-25, affinitychromatography on Heparin-Sepharose CL-6B and Concanavalin-A-Sepharose4B. The final purification of EPF was achieved by high performancehydrophobic interaction chromatography, followed by three reversed phase(RP)-HPLC steps. After the final RP-HPLC step, EPF was isolated assingle UV absorbing peak coincident with biological activity, wellseparated from a number of minor contaminants. The biological andradioactivity of an iodinated sample of this material eluted withidentical retention time when fractionated under the same conditions.When analysed by SDS-PAGE and visualised by autoradiography, theiodinated material ran as a single band of approximate Mr 10,000, againcoincident with biological activity. The approximate yield of EPF bythis method was 5 μg per 100 platelet units.

This demonstrates that it was necessary to use this complex purificationprocedure to obtain only a small amount of native EPF and thus thismethod could not be used on a commercial scale. In this regard, the onlypractical sources known for obtaining native EPF at this time wereplatelets and regenerating liver.

Surprisingly, in accordance with the present invention, the finalfractionated EPF when subjected to sequencing as more fully describedhereinafter found that the structure of native EPF corresponded tochaperonin 10 which could not have been predicted from theaforementioned prior art.

This unexpected discovery as will be apparent from the disclosurehereinafter has now been reduced to practice in that antibodies torecombinant chaperonin 10, as described hereinafter as well asderivatives or fragments thereof, has been found to have all thebiological activity previously associated with antibodies to EPF. EPFcan now be produced commercially which was not the case previously usingsuitable techniques for producing recombinant cpn10 or producing cpn10synthetically. It will thus be apparent that this will also facilitatecommercial production of antibodies to cpn10.

SUMMARY OF THE INVENTION

In one aspect, the invention provides for the production of antibodiesspecific for recombinant cpn10 having the amino acid sequence ashereinafter described as well as derivatives or fragments thereof.

Another aspect of the invention is the use of such antibodies. The useof the antibodies includes cellular growth suppressing activity or animmune enhancing activity.

The present invention includes within its scope the use of monoclonaland/or polyclonal antibodies to the recombinant cpn10 as hereinafterdescribed as well as fragments or derivatives thereof.

EXPERIMENTAL A. Purification of cpn10 and Antibody Production (i)Purification of Human EPF from Human Blood Platelets (FIGS. 1a, 1b, 1c,1d)

Extraction

Platelet concentrates (from the Blood Bank), up to 7 days clinicallyoutdated, were washed with Tyrodes buffer, following the techniquesdescribed in Methods in Enzymology, 1989, 169 7-11, snap frozen inliquid N₂ and stored at -70° C.

Immediately prior to purification, approximately 100 washed plateletunits were thawed in a boiling water bath, then held at 75-85° C. for 15min with continuous, gentle stirring. After cooling on ice, cellulardebris was removed by centrifugation (8000 g, 20 min, 4° C.) and thepellet extracted twice by homogenisation in 0.05 M-acetic acid/0.1M-NaCl/0.1 mg/ml sodium azide pH 3.0 followed by centrifugation (8 000g, 15 min 4° C.). The three supernatants were pooled giving a totalextract volume of 500-600 ml.

Ion-exchange chromatography

This extract from 100 platelet units was adjusted to pH 3.0 with conc.HCl and stirred gently, overnight, 4° C., with 250 ml SP-Sephadex C-25(Pharmacia-LKB), previously swollen and equilibrated with 0.05 M-aceticacid/0.1 M-NaCl pH 3.0. The gel was then packed into a column washedwith 20 vol of the same buffer and eluted with 5 vol 0.5 M-sodiumphosphate buffer/0.05 M-NaCl pH 7.5. The gel was then discarded.

Affinity chromatography

The SP-Sephadex eluate was adjusted to pH 6.3-6.4 with conc. HCl andapplied to a column of Heparin-Sepharose CL-6B (2.5×7.5 cm;Pharmacia-LKB) previously equilibrated with 0.05 M-sodium phosphatebuffer/0.05 M-NaCl pH 6.3. The column was then washed with 5 vol of thesame buffer and eluted with 5 vol 0.05 M-Tris-HCl/5 mM-CaCl₂ /0.2 M-NaClpH 7.5, applied in the reverse direction to that used for sampleapplication.

High performance hydrophobic interaction chromatography (HIC-h.p.l.c.)

Solid (NH₄)₂ SO₄ was added to the Heparin-Sepharose eluate to a finalconcentration of 2 M and, after passage through an 0.45 μm filter, thesample was pumped through a dedicated solvent line onto a TSK Phenyl 5PWcolumn (7.5×75 mm, Pharmacia-LKB), previously equilibrated with 0.1M-Tris-HCl pH 7.0/5 mM CaCl₂ /2 M-(NH₄)₂ SO₄. The column was washed with10 vol of the same buffer and eluted with a 50 min linear gradient fromthis buffer to 0.1 M-Tris-HCl pH 7.0/5 mM-CaCl₂ /10% acetonitrile. (FIG.1a)

RP-h.p.l.c.-1

Active HIC-h.p.l.c. fractions were pooled, then fractionated on a C₃column (Ultrapore RPSC, Beckman Instruments) using a solvent systemconsisting of A, 0.04 M Tris/HCl pH 7.0/5 mM-CaCl₂ and B, 0.04M-Tris/HCl pH 7.0/5 mM-CaCl₂ /80% (v/v) acetonitrile. The column wasequilibrated with Solvent A prior to sample application, after which itwas washed with 5 vol solvent A and eluted with a 30 min linear gradientfrom this solvent to 75% solvent B. (FIG. 1b)

RP-h.p.l.c.2

Active fractions from RP-h.p.l.c.-1 of several 100 unit plateletpreparations were pooled, EDTA and DTT added to a final concentration of20 mM and 1 mM respectively and the mixture allowed to stand for 0.5-1h, 4° C. Following dilution with 2 vol solvent A, it was applied to a C₃column, dedicated to this and subsequent steps, and fractionated asdescribed for RP-h.p.l.c.-1, but omitting CaCl₂. (FIG. 1c)

Rph.p.l.c.3

Active fractions from RP-h.p.l.c.-2 were pooled, trifluoroacetic acid(TFA) added to a final concentration of 0.1% and, following dilutionwith 2 vol 0.1% TFA, the mixture was applied to the C₃ column, which hadbeen equilibrated previously with 0.1% TFA. The column was then elutedwith a 30 min linear gradient from this solvent to 60% (v/v)acetonitrile/0.1% TFA, followed by a 3 min linear gradient to 90% (v/v)acetonitrile/0.1% TFA. Active fractions were pooled. (FIG. 1d)

One unit represents platelets from a single blood donation which isapproximately 500 ml. The "active fractions" were fractions active inthe rosette inhibition test.

Purification of EPF from other sources

The purification of cpn10 from various sources have been summarised inCavanagh et al., 1994, Eur. J. Biochem. 222 551-560.

Biological activity followed the same pattern throughout the complexpurification scheme described above for human platelets. Furthermore thefinal active fraction from all sources was bound specifically by animmobilised monoclonal anti-EPF and could be recovered virturallyquantitatively (see FIG. 1e).

These studies are important for several reasons:

A. The biochemical and immunological similarity observed with all thesematerials provides strong evidence that the bioassay is detecting asingle substance or closely related family of substances acting indiverse biological situations.

B. The active agents purified from all of these materials are fromseveral to many orders of magnitude more potent than virtually all ofthe substances previously reported to be EPF summarised in theabovementioned Morton et al., 1992 reference. This confirms our surmise,based on detailed analysis of the EPF bioassay as discussed above, thatactivity associated with most putative EPF preparations must reflect thepresence of a very minor contaminant.

C. The only source materials providing sufficient EPF to study at theprotein (as opposed to activity) level were platelets and regeneratingliver, yielding, respectively, an average of 15 μg per 100 units(equivalent to ˜50 liter blood) and 5 μg per 40 g tissue (liver remnantfrom 6 rats). It is immediately apparent that far more EPF is presentwithin the cell than appears in the extracellular space: nevertheless,accumulated knowledge of the biology of EPF (reviewed recently in theabovementioned Morton et al. 1992 reference) indicates that thisextracellular appearance is not fortuitous.

Human platelet-derived EPF, being most abundant, has been studied insome detail. On SDS-PAGE, it ran as a single band of M_(r) approx, 8500, coincident with biological activity (see FIG. 2a); EPF fromregenerating rat liver exhibited identical behaviour. Mass spectometryof the platelet material provided an accurate and precise determinationof molecular mass 10 843.5±2 Da, along with definitive evidence of thehigh degree of homogeneity of the preparation (see FIG. 2b). Followingattempts at Edman degradation, which indicated that the molecule isN-blocked, proteolytic cleavage of approx. 4 nmol EPF was undertaken.Resultant peptide fragments were separated by reversed-phase HPLC andsubjected to sequencing by Edman degradation. Three areas of sequencecontaining 12 (fragment 1, SEQ ID NO:22), 27 (fragment 2, SEQ ID NO:23)and 33 (fragment 3, SEQ ID NO:24) residues were found to correspond withresidues 7 to 18, 27-53 and 69-101 (the C-terminus) in rat mitochondrialcpn10. In fragment 2, residue 52 was different (S in cpn10, G in ratcpn10; this change alone could account for human cpn10 being 30 Dalarger than rat cpn10). All other residues were identical, consistentwith the highly conserved nature of chaperonins (see FIG. 2c).

Since confirming sequence identity between EPF and cpn10, severalstudies of functional relationship have been performed, using ratmitochondrial cpn10. E. coli cpn10 (known as groES) and E. coli cpn60(groEL). First it has been demonstrated that cpn10 can act as EPF. Ratcpn10 was tested in the EPF bioassay and found to be positive over therange of dilutions expected; this activity could be neutralised bymonoclonal antibodies to EPF. Interestingly, E. coli cpn10, which is˜40% homologous with rat cpn10, exhibited no activity in the bioassay.This is consistent with the observation that E. coli conditioned mediumis not active in the EPF bioassay, while medium conditioned by allmammalian cell lines tested, as well as by yeast cells is active. Cpn60was inactive in the bioassay and had no effect upon the activity of EPF.It was then shown that EPF can act as cpn10. EPF was mixed with cpn60,in the presence or absence of ATP, and the mixture fractionated on a TSKG3000SW gel permeation column; resultant fractions were analysed bySDS-PAGE. Cpn60 is a decatetramer and elutes in the excluded volume ofthis column (exclusion limit 300 000). In the presence of ATP, but notin its absence, EPF also appears in this fraction, demonstratingformation of a stable complex with cpn60. This fraction was active inthe EPF bioassay but the equivalent fraction from the experiment withoutATP (where EPF did not associate with cpn60) was not (see FIG. 3a). ThusEPF and cpn10 activity reside in the same molecule.

These investigations provide unequivocal evidence that platelet-derivedEPF is a structural and functional homologue of cpn10; the relationshipbetween cpn10 and activity in the rosette inhibition test was thenexamined (FIG. 3b). In the presence, but not in the absence of ATP,immobilised cpn60 could remove all activity from the archetypal sourcematerial, pregnancy serum and activity could be recovered by removingATP from the immobilised complex. As with the experiment described inFIG. 3a, this requirement for ATP demonstrates the specificity of theinteraction between cpn60 and the active moiety; cpn10 is thus themolecular entity initiating response in the EPF bioassay.

Identification of EPF as a cpn10 has been a major step forward inresearch on this subject and helps to explain many of the findings thathave been made to date. Criticism has been raised against claims the EPFproduction occurs in such a wide variety of biological situations e.g.pre-and post-implantation pregnancy, primary and tumour cellproliferation and platelet activation. In its role as a hsp (heat stressprotein) following the advent of the present invention, these are allconditions in which the rapid onset of EPF production would now beexpected. Functions of hsp's that are vital to the survival of cells areintracellular as shown in the Linquist et al. reference above. Incontrast, the activity of EPF described to data is extracellular; forexample, it appears in serum of mice within 4 to 6 hours after mating asdiscussed in Morton et al., 1987, Current Topics in Development Biology,Vol 23 73-92 and 4 to 8 hours after partial hepatectomy in rats as shownin the Quinn PhD thesis (1991). We have shown that EPF can act in anautocrine mode as discussed in the Quinn et al., 1990 reference referredto above or exocrine mode as discussed in the Rolfe et al. 1988 referredto above: these are not roles previously described for hsp's

It will also be appreciated that since the structure of EPF is nowknown, it can be produced in commercial quantities by any suitabletechnique such as by recombinant DNA techniques or by chemicalsynthesis.

(ii) Production of Anti-cpn10-Derived Peptide

Described here are the methods used and results encountered in theproduction of anti-cpn10-derived peptide. Peptides of cpn10 may includethe following amino acid sequences (SEQ ID NOS:1-12):

(i) AGQAFRKFLPL;

(ii) Ac-AGQAFRKFLPL;

(iii) EKSQGKVLQAT

(iv) A₁ AGQAFRKFLPLA₂ ;

(v) AGQAFRKFLPLA₂ ;

(vi) A₁ AGQAFRKFLPL;

(vii) Ac-A₁ AGQAFRKFLPLA₂ ;

(viii) Ac-AGQAFRKFLPLA₂ ;

(ix) Ac-A₁ AGQAFRKFLPL;

(x) A₁ EKSQGKVLQATA₂ ;

(xi) EKSQGKVLQATA₂ ;

(xii) A₁ EKSQGKVLQAT;

wherein A₁ and A₂ are amino acid sequences which may be added to one oreach end of molecules (i) through (xii) and Ac is acetyl.

Anti-cpn10-derived peptides antibodies may include antibodies raisedagainst any one of the aforementioned amino acid sequences (i)-(xii). Asan example, antibodies have been raised against an N-terminal fragment(Ac-AGQAFRKFLPLC, SEQ ID NO:130 and an internal fragment (EKSQGKVLQATC,SEQ ID NO:14).

It will be appreciated in the abovementioned peptides that such peptidesmay include a single amino acid addition, deletion or substitution andthe invention also includes antibodies raised against such peptides.

Methods

Synthesis of cpn10 derived peptides

Peptides were synthesized to correspond with an N-terminal fragment(N-peptide i.e. Ac-AGQAFRKFLPLC, SEQ ID NO:13) and an internal fragment(I-peptide i.e. EKSQGKVLQATC, SEQ ID NO:14).

Conjugation of peptides to ovalbumin

Peptides were conjugated to ovalbumin by the hetero-bifunctional reagentSPDP, following manufacturer's instructions (Pharmacia-LKBBiotechnology, Uppsala, Sweden).

Immunisation schedules

Adult outbred New Zealand rabbits were immunised with one of theconjugates in 4×weekly injections followed by several monthly boosts.

For injection, the antigen was dialysed into 0.9% saline (Mr 12-15000cut off dialysis tubing, Visking, Union Carbide, Ill., USA) andemulsified with an equal volume of Freund's adjuvant (complete for thefirst injection, incomplete thereafter). Immunisations were via the s.c.route. Table 1 shows the amount of antigen injected.

Screening of antiserum

Antisera were tested in an ELISA against the relevant antigens (viz.I-peptide or N-peptide; ovalbumin) (5 mg/ml). Bound IgG was detected bythe biotin-streptavidin system (Amersham) with o-phenylene diamine assubstrate. Absorbance was read at 492 nm.

Anti-N-peptide Abs were also tested in parallel with anti-EPF Abs #810and ·816 (Athanasis-Platsis et al., 1989, J. Reprod. Fert. 87 495-502)against platelet derived EPF (1 mg/ml) (Cavanagh et al., 1994, Eur. J.Biochem. 222 551-560) and N-peptide (5 mg/ml).

Purification of antibodies

IgG was purified from serum by affinity chromatography. N and Ipeptides, and ovalbumin were coupled separately to a HiTrap™ affinitycolumn (HiTrap NHS-activated 1 ml, Pharmacia-LKB) following themanufacturer's instructions. Each column was equilibrated with 0.05NaPi-0.5M NaCl, pH 7.4, and the relevant antiserum applied, according tothe manufacturer's instructions. After extensive washing withequilibration buffer, bound rabbit IgG was eluted by 0.2M glycine, pH2.5. The pH of the eluate was adjusted with 2M Tris to approximately7.4.

The purity of the Abs in the eluted fractions was determined bySDS-PAGE, then the strongest fractions pooled.

Protein estimation

Protein (IgG) was determined by the method of Lowry (Lowry et al., 1951,J. Biol. Chem. 193 265-275), using a commercial preparation of Folin andCiocalteu's reagent (Stansens, Qld, Australia). The strandard curve wasconstructed with a purified rabbit IgG preparation (20 mg/ml; Silenus,Howthorne, Australia).

Results

The ELISA screening of the antibodies provided some interesting results.

The anti-peptides Abs tire decreased even with repeated boosting (FIGS.4a, 4b), while the production of anti-ovalbumin control Abs gave anormal response (FIG. 4c). Note that the titre of anti-ovalbumin Abs inrabbits immunised with the peptide conjugates (FIGS. 4a, 4b) decreasedas well.

Cross reactivity studies are shown in FIG. 5.

The "anti-EPF #810" and "anti-EPF #816" antibodies are typical prior artanti-EPF antibodies disclosed in Athanasis-Platsis et al., (1989),Supra. Such prior art antibodies being IgG polyclonel antibodies are lowaffinity antibodies as shown in FIG. 5 when compared to high affinityantibodies of the invention exemplified by the anti-N peptide antibody.

The titre of affinity purified Abs was determined by ELISA against theimmunising peptide conjugated to bovine serum albumin (BSA). This testalso demonstrated the efficiency of the procedure. Results are shown inTable 2.

These Ab preparations where shown in SDS-polyacrylamide gelelectrophoresis to be approximately 95% pure.

Conclusion

The decreasing titre of Abs during the immunising schedule suggests arole for cpn10 in the proliferation of B cell clones. The instability ofantibody-producing B cell clones which produce anti-EPF antibodies hasbeen previously described. The pattern of anti-EPF antibody productionwas as described above, with maximum titres obtained 5 weeks after theinitial immunisation and then falling despite frequent boosting. Invitro, hybridomas producing anti-EPF antibodies were inherently unstable(Quinn et al., 1990, Clin. Exp. Immunol. 80 100-108). The difficulty inmaking a stable cell line of a hybridoma which produces ananti-EPF/cpn10 antibody may be due to the autocrine action of EPF/cpn10in cell proliferation, i.e. antibodies neutralize EPF/cpn10 whichproliferating cells produce for their own growth advantage.

(iii) Preparation of Antibodies to Recominbant cpn10

Cloning of human cDNA encoding cpn10 and production of cpn10

Production for commercial use may be obtained by inserting a mammaliancpn10 gene, preferably a human cDNA cpn10 gene, into a suitable vectorsuch as plasmids from the pGEX system, and pET system expressing theencoded mammalian cpn10 and purifying the recombinant cpn10.

    ______________________________________                                        Abbreviations                                                                 ______________________________________                                        ANGIS  Australian National Genomic Information Service                        bp     base pair                                                              BSA    bovine serum albumin                                                   cDNA   complementary DNA                                                      cpn10  Chaperonin 10                                                          DNA    deoxyribonucleic acid                                                  E. coli                                                                              Escherichia coli                                                       GSH    glutathione (reduced form)                                             GST    glutathione-S-transferase                                              LB     Luria-Bertani Broth                                                    M      Molar                                                                  ORF    open reading frame                                                     PCR    polymerase chain reaction                                              rEPF   recombinant Early Pregnancy Factor                                     RSP    reverse sequencing primer                                              SDS    sodium dodecyl sulphate                                                SDS-PGE                                                                              sodium dodecyl sulphate-polyacrylamide gel electrophoresis             Tris   Tris(hydroxymethyl)aminomethane                                        USP    universal sequencing primer                                            ______________________________________                                    

Materials and Methods

Cloning of Human cpn10 Open Reading Frame

Polymerase chain reaction (PCR) was used to initially amplify part ofthe ORF (274 bp) of the human cpn10 cDNA from a melanoma cell line A2058cDNA lambda library (Stratagene). A degenerate cpn10 amplimer (P1) wasdesigned from the amino acid sequence VLDDKDYFL (SEQ ID NO:15)corresponding to amino acid residues 83-91 of human cpn10. The primer P1has the sequence 5' ARRAARTARTCYTTRTCRTC 3' (SEQ ID NO:16) where R is Aor G and Y is C or T. The reverse sequencing primer (RSP) was used forPCR amplification (the non-specific primer) as well as for sequencingDNA constructs and has the sequence 5' CAGGAAACAGCTATGAC 3' (SEQ IDNO:17). The universal sequencing primer has the sequence 5'GTAAAACGACGGCCAGT 3' (SEQ ID NO:18). PCR amplification of the phagelibrary was achieved using a non-specific upstream amplimer (RSP) andP1, each at 0.5 μM final concentration, 1.5 mM MgCl₂ (PharmaciaBiotech), 1X polymerase buffer (Boehringer Mannheim) and 5 units ofThermus aquaticus DNA polymerase (Boehringer Mannheim) in a final volumeof 50 μL. For 30 cycles, the parameters were: denaturation at 94° C. for1 min, annealing at 40° C. for 30 sec and extension at 72° C. for 3 min.A final extension at 72° C. for 7 min was followed by a soak cycle at 4°C. for 10 min. An aliquot of 1 μL was reamplified under the sameconditions to increase the copy number.

Two cpn10 specific amplimers encompassing the open reading frame weredesigned. The upstream primer P2, 5'-GCGCGGATCCATGGCAGGACAAGCGTTTAG-3'(SEQ ID NO:19), was designed from the sequence of the intitial PCRfragment. The downstream primer P3, 5'-ATATGAATTCAGTCTACGTACTTTCC-3'(SEQ ID NO:20) was designed from sequence obtained from the ExpressedSequence Tag database via ANGIS (Accession No. HUM00TB037). A 319 bpfragment was amplified from the phage library using the same reactionand cycling conditions as above except the annealing temperature was 50°C.

DNA Constructs and Analysis

All restriction enzyme digests of PCR products and vectors wereperformed according to Sambrook et al. (Sambrook et al., 1989, MolecularCloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Press, ColdSpring Harbor, N.Y.) using restriction enzymes and their buffersobtained from boehringer Mannheim. The initial PCR fragment was digestedwith Eco R1 and ligated (Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual. 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor,N.Y.) into the Eco R1 and Sma I sites of pBluescript KS(+) (Stratagene)creating the plasmid pRM1 (FIG. 6a; partial cpn10 insert 274 bp). The319 bp product was digested with Bam HI and Eco R1 and initially clonedinto the expression plasmid pGEX-2T (Pharmacia Biotech) creating theplasmid pRM2 (FIG. 6b). To confirm its identity, the Bam HI-Eco R1fragment was subcloned into pBluescript (SK+) (pRM3; FIG. 6c) andsequenced. DNA was analysed on 0.8-1.0% (w/v) agarose gels containingethidium bromide and after electrophoresis was viewed under UVillumination.

Transformation of E. coli

Competent E. coli DH5α cells (100 μL) were transformed with the plasmidsby the heat pulse method (Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual. 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor,N.Y.). The mixture of cells and DNA (10-100 ng) was placed on ice for 30min and heat pulsed for exactly 2 min at 42° C. and placed back on icefor 2 min. The cells were allowed to recover at 37° C. with shaking for1 hr after the addition of 0.9 mL of LB. A 100 μL aliquot was platedonto LB agar plates supplemented with Ampicillin at a finalconcentration of 100 μg/mL. After incubation overnight at 37° C., randomcolonies were selected for further investigation.

DNA sequence determination

Restriction fragments of the PCR products were cloned into pBluescriptand sequenced in both orientations by the dideoxy chain-terminationmethod using the T7 Polymerase Kit according to the manufacturer'sinstructions (Pharmacia Biotech). Approximately 2 μg of plasmid DNA wasdenatured, ethanol precipitated and annealed to either the USP, RSP orP3. The sequencing reactions were electrophoresed on a 8% acrylamide/46%urea gel. After fixing and drying, X-ray film was exposed to the gelovernight and developed.

Expression and purification of recombinant cpn10 in E. coli

Clones transformed with pRM2 were screened for expression of theGlutathione-S-transferase fusion protein on a small culture scale (2 ml)according to methods described by Smith et al. (Smith et al., 1988, Gene67 (1) 31-40). An overnight culture was diluted, induced to express thefusion protein by the addition of IPTG to 0.1 mM and grown at 37° C. forseveral hours. The cells were pelleted, lysed in PBS/0.1% Triton X-100and the lysate mixed with 50% Glutathione-Agarose beads (Sigma ChemicalCompany). The recombinant fusion protein was eluted from the affinitybeads by boiling in SDS loading buffer. An aliquot of the sample was runon a 10% SDS-PAGE gel. The gel was fixed and then stained with Coomassieblue. After confirming the expression of the fusion protein thepurification of rcpn10 from the GST moiety was undertaken on a largerscale.

Cells were grown and induced as above, the cell pellet resuspended inPBS, sonicated (output level 4, 50% duty cycle, 2×30 sec) and the celllysate stored at -30° C. Lysate from 10 liter cell culture was thawedand rcpn10 isolated by similar techniques to those used by Gearing etal. (Gearing et al., 1989, Biotechnology 7 1157-1161) for isolation ofrLIF. Briefly, Triton X-100 was added to a final concentration of 0.1%and cellular debris removed by centrifugation (15 min, 15000 rpm, 4°C.). Ten ml glutathione-Sepharose 4 B gel (Pharmacia--LKB Biotechnlogy)was added to the supernatant and the slurry mixed for 2 hr, 4° C. Thegel was pelleted, washed×5 with 50 ml PBS/0.1% Triton X-100 once with 50ml 0.05 M Tris-HCl pH 8.0/0.15 M NaCl and once with 0.05 M Tris-HCl pH8.0/0.15 M NaCl/2.5 mM CaCl₂. The gel was resuspended in 4 ml of 0.05 MTris-HCl pH 8.0/0.15 M NaCl/2.5 mM CaCl₂ buffer, 1000 units thrombin(Sigma T6884) added and the slurry was mixed in a shaking waterbath for1 hr, 37° C. The gel was pelleted, the supernatant retained, and the gelwas then washed with 3×4 ml 0.05 M Tris-HCl pH 8.0/0.15 M NaCl. Thesewashes and the first supernatant, which contain the rcpn10, were pooled,yielding 4-5 mg recombinant protein. Additional rcpn10, which wasnon-specifically bound to the gel, was recovered as follows. Four ml0.05 M Tris-HCl pH 8.0/2 M NaCl was added and the slurry mixed for 2 hr,4° C.

After pelleting, the gel was washed with 3×2 ml of this 0.05 M Tris-HClpH 8.0/2 M NaCl buffer, the washes pooled with the first supernatant,yielding a further approximately 1 mg rcpn10. Protein concentrationswere estimated by the method of Lowry et al. (Lowry et al., 1951, J.Biol. Chem. 193 265-275); proteins were analysed by SDS-PAGE using 15%Tris-Tricine gels (Schagger et al., 1987, Anal. Biochem. 166 368-379).

The recombinant cpn10 has two additional amino acids at the N terminus.The N terminus of the recombinant protein is Gly-Ser-Methionine-Alawhereas the N-terminus of native protein is Ac-Ala. The amino acidsequence (SEQ ID NO:21) of the recombinant cpn10 is as follows:GSMAGQAFRKFLPLFDRVLVERSAAETVTKGGIMLPEKSQGKVLQATVVAVGSGSKGKGGEIQPVSVKVGDKVLLPEYGGTKVVLDDKDYFLFRD GDILGKYVD

Antibodies were raised against the GST:rcpn10 fusion protein.

Antibodies against the recombinant protein were raised in rabbits usingthe same schedule described for producing anti-peptide antibodies.Approximately 10 μg protein was used for each injection. Rabbit serumwas screened for anti-cpn10 antibodies by ELISA, using the techniquedescribed for screening anti-peptide antibodies with the exception thatplates were coated initially with cpn10 (5 μg/ml). The antibody (Ab)titres against cpn10 and against the whole fusion protein (in this case,GST:rcpn10, 5 μg/ml, was bound to the plate) in serum of rabbit #42 areshown in FIG. 7. Titration of a serum sample against cpn10, taken fromthis rabbit after the 4th booster does, is illustrated in FIG. 8.

B. Pregnancy Termination

In another aspect of the invention, pregnancy may be terminated byadministration of antibodies specific for cpn10 to a pregnant subject.The antibodies may be raised against cpn10 or derivatives therefrom. Theadministration of these antibodies preferably occurs at thepre-implantation stage (1-2 cell stage) or at the peri-implantationstage. Pregnancy termination with anti-cpn10 antibodies is demonstratedbelow by way of example in a mouse model system. The mouse model systemis by way of example only and the method is not limited to mice. Themethod may be applied to any suitable mammalian species including man.

(i) Antibodies Raised Against cpn10 Peptides Terminates Pregnancy atPre-Implantation Stage

Anti-cpn10 antibodies

The preparation and characterisation of these antibodies have beendescribed. In these experiments, antibodies used were those preparedagainst the N-terminal peptide (cpnN) and an internal peptide (cpnI);cpnN and cpnI are active in the rosette inhibition test. IgG wasprecipitated from anti-serum by 45% ammonium sulphate and theconcentration determined by Lowry and gel electrophoresis. The IgGpreparations were tested in an ELISA against the immunising peptide,conjugated to bovine serum albumin. The preparations were also testedfor their ability to neutralise activity in mouse pregnancy serum.Various concentrations of antibody were incubated with an equal volumeof mouse serum then the mixtures tested for activity in the rosetteinhibition test. The lowest concentration of antibody that couldcompletely neutralise EPF activity was determined (see Cavanagh et al.,1994, Eur. J. Biochem. 222 551-560). Ten pg anti-N-peptide Abneutralised the activity of 1 ml of pregnancy serum while 4 nganti-I-peptide was needed for complete neutralisation.

Passive Immunisation

Mature outbred male and female Quackenbush mice were caged in pairs at7:30 a.m. and separated at 8:30 a.m. Female mice with vaginal plugs wereinjected with anti-N-peptide/ovalbumin, anti-I-peptide/ovalbumin oranti-ovalbumin IgG preparations at 9:00 a.m. and 5:00 p.m. on days 1(day of mating) and 2 of pregnancy. The dose of specific IgG injected inthe 2 dose regimen was estimated as approximately 1 mg/mouse/day. On day7, mice were euthanased with CO₂, uteri examined for implanted embryosand the number of corpora lutea (CL) counted. In each group, the numberof embryos/CL in the mice treated with the test IgG was compared withthe number receiving the same dose of control IgG (χ² test).

Results

The results, shown in Table 3, clearly demonstrate that neutralisationof activity in pregnancy serum can adversely affect embryonic viabilityin the early stages of pregnancy. The ability of antibodies toneutralise cpn10 activity in the rosette inhibition test is an in vitromonitor of their ability in vivo to adversely affect pregnancy.

C. Cancers and Tumours

A further aspect of the invention is the suppression of growth ofabnormal cells by the administration of antagonists of cpn10 to asubject. Said abnormal cells or aberrant growth of normal cells includetumour or cancer cells; aberrant growth of normal cells includesdiseases such as in psoriasis or Reiter's syndrome. Tumour cells includethose from both benign and malignant growths. Cells from malignantdiseases such as solid tumours and haematological cancers may also beincluded. An example of the suppressing effect of tumour cell growth isdemonstrated by experiments with murine B16 melanoma and MCA-2fibrosarcoma cell lines.

(i) Effect of Anti-cpn10-Derived Peptides Antibodies (ABS) on the Growthof Tumour Cells in vitro

Introduction

The following studies investigate the possibility that cpn10, producedby tumour cells in vitro, is also required by these cells for theircontinued growth.

Methods

Cell culture

Cell lines were cultured under standard conditions in basal medium.Dulbecco's modification of Eagle's medium (DMEM; ICN BiochemicalsAustralasia Pty. Ltd., Australia), supplemented with 10% foetal calfserum (FCS, ICN) 20 mM glutamine (ICN) and antibiotics [100 μg/mlstreptomycin (ICN), 100 U/ml penicillin (CSL, Melbourne, Australia)], at37° C. in a humidified atmosphere of 5% CO₂ in air.

Cells were maintained in the logarithmic (log) phase of growth.Monolayers were dissociated, after washing in serum-free medium, by ashort exposure, at 37° C., to a solution of 0.1% w/v Trypsin and 0.02%w/v versene in calcium and magnesium free balanced salt solution. Theaction of the trypsin was neutralised with the addition of mediumcontaining 2% v/v FCS and the cells were recovered by centrifuging 200 gfor 5 min, washed a further two times in serum free medium, after whichthey were seeded into culture dishes or 96 well plates (NUNC).

Stocks of cell lines were maintained frozen in liquid N₂ at all times.

Preparation of anti-peptides Abs for co-culture experiments

Affinity purified anti-N Abs, anti-I Abs and anti-ovalbumin Abs (controlantibodies) were exchanged into DMEM and adjusted to a finalconcentration of 1 mg/ml. The preparations were sterilised by passagethrough a 0.2 μM cut-off filter (Minisart, Sartorius Gmbh, Goettingen,Germany). As a control medium, DMEM alone was similarly treated.

Co-culture of tumour cells with anti-peptides Abs

The murine B16 melanoma and MCA-2 fibrosarcoma cell lines were studied.The cells (10³) were seeded in triplicate, in 0.2 ml culture medium(DEMEM+10% FCS (heat inactivated) containing doses of anti-peptide Abs,or control Ab, in the range 62.5-500 μg Ab/ml (final concentration).Cells were similarly seeded into filtered medium containing no antibody.Cultures were examined after a 96 h culture period. Viability wasassessed by trypan blue exclusion and uptake of methyl-[³ H]thymidine5'-triphosphate ([³ H]thymidine; Amersham International, Amersham, UK)was used to monitor the rate of cell division. Relative [³ H]-thymidineuptake for each antibody dose was calculated by expressing the mean cpmincorporated (from triplicate wells) as a percentage of the average cpmincorporated in the wells containing no antibody.

Determination of cell viability

Cells dissociated by trypsin, were mixed with an equal volume of 0.1%w/v trypan blue in PBS and spread onto a haemocytometer. Cell viabilitywas calculated as the percentage of cells excluding the dye.

Determination of [³ H]Thymidine uptake

After 80 hours incubation, cells were then cultured for a further 16hours with 0.5 μCi [³ H]thymidine per well. After incubation thesupernatant medium of adherent cells was removed and each well waswashed twice with warm DMEM. Acid precipitable material was separated byaddition of 250 μl ice cold 5% w/v trichloroacetic acid (TCA, BDHChemicals, Australia Pty Ltd. Kilsyth, Victoria, Australia) to each well(Plate, 1974, J. Exp. Med. 139 851-861). The precipitate was washedtwice with TCA and solubilized in 0.3 ml 0.25 N NaOH; 250 μl of thispreparation was mixed with 2 ml scintillation cocktail (Emulsifier safe,Packard Instruments Co., Meriden, Conn., USA) and cpm incorporated intoacid precipitable material were determined for each well by β-counting.

Immunocytochemistry

Human T-cell leukaemia cells Molt 4 (ATCC CRL 1582) were maintained inlog phase in RPMI+10% FCS. Cells were washed three times in PRMI+FCS andincubated (10⁶ cells) with 10 μg (in 0.1 ml) affinity purified anti-Npeptide Ab, anti-I peptide Ab or control antibody (anti-ovalbumin Ab).Control tests contained 10⁶ normal spleen cells. Bound antibody wasdetected by anti-rabbit biotinylated IgG, F(ab'₂) fragment (Amersham),followed by streptavidin-fluorescein according to the manufacturer'sinstructions. Binding was visualized by UV microscopy.

Results

Tumour cell growth is perturbed by co-culture with anti-cpn10-derivedpeptides Abs.

Incubation of B16 melanoma and MCA-2 fibro sarcoma cells in increasingconcentrations of anti-peptide Abs resulted in a significant decrease ofcell division and increased cell death after 96 h incubation (FIGS. 9a,9b; 10a, 10b). Incubation of cells in similar concentrations of controlAb had no effect (FIGS. 9c, 10c).

Anti-I peptide antibody bound to cpn10 in the surface of Molt 4 cells.No binding was detected with anti-N peptide or anti-ovalbumin antibodyon Molt 4 cells or with any of the abovementioned antibodies on normalspleen cells. This is the first visualization of extracellular cpn10(FIG. 11).

Conclusion

The studies described here have established that anti-cpn-10-derivedpeptides Abs inhibit the growth of tumour cells. The anti-proliferativeeffect of culturing B16 and MCA-2 cell lines in increasing doses ofanti-peptides Abs is evidence that the growth of these cells isdependent upon continued presence of cpn10. These studies haveestablished that the tumour cells require cpn10 for proliferation invitro.

OTHER ASPECTS OF THE INVENTION

The above mentioned N-terminal fragment and internal fragment areregions of the molecule which are active in the rosette inhibition testand therefore function as active centres.

Pharmacological antagonists can be constructed, using conventionalmeans, by modification of the structure of these active centres, so thatbinding to target sites, e.g. tumour cells, may occur without targetactivation. By interfering with the action of the whole cpn10 moleculeon tumour cells, these antagonists will mimic the anti-proliferativeeffect described above for anti-cpn10 antibodies.

The invention also includes within its scope an assay for measuringanti-cpn10 antibody in a sample including the steps of:

(1) reacting substantially purified cpn10 with the sample; and

(2) determination of the amount of anti-cpn10 antibody in the sample bydeterming the binding between the antibody and cpn10.

It will also be appreciated from the foregoing that data is available(e.g. Quinn et al., 1992, Cancer Immunol. Immunother. 34 265-271) thatanti-EPF antibodies are useful in suppression of tumour growth in amouse model. Such data supports the assertion that anti-EPF antibodieswill suppress tumour growth in vivo or in vitro.

The dosages utlised in the administration of antagonists or antibodiesare in the range of 1-1000 (more preferably 50-200) μg/kg of body weightfor antagonists and 1-1000 (more preferably 50-200) mg/kg of body weightfor antibodies. These dosages are based on a molecule which has the samemolecular weight as cpn10 and dosages should be adjusted accordingly.

                  TABLE 1                                                         ______________________________________                                                      Dose                                                            Ag            (μg/injection)                                               ______________________________________                                        I-peptide     200                                                             N-peptide     400                                                             Ovalbumin     500                                                             ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                                    TITRE                                                                         1 log.sub.2 reciprocal serm dilution                                                Pre                                                                           Column            Unbound                                           Test      (serum     Bound  (serum                                    Anti-serum                                                                            antigen   before column)                                                                           (fractions)                                                                          after column)                             ______________________________________                                        Anti-N  N-peptide 15         18     11                                        ovalbumin                                                                     Anti-   I-peptide 13         19     10                                        ovalbumin                                                                     Anti-   Ovalbumin 19         25     >17                                       ovalbumin                                                                     ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                                          Corpora                                                     Antibody                                                                              No. of    lutea/     Embryo/                                          (total dose                                                                           animals   mouse      mouse                                            2 mg/mouse)                                                                           in group  (mean ± sem)                                                                          (mean ± sem)                                                                          p*                                    ______________________________________                                        Anti-N- 6         19.1 ± 1.2                                                                            10.6 ± 3.8                                                                            <0.05                                 peptide-                                                                      ovalbumin                                                                     Anti-I- 6         20.8 ± 0.8                                                                            17.1 ± 1.1                                                                            <0.02                                 peptide-                                                                      ovalbumin                                                                     Anti-   5         17.8 ± 1.0                                                                            16.8 ± 0.5                                                                            NS                                    ovalbumin                                                                     ______________________________________                                    

TABLE LEGENDS Table 1

Dose of antigen per injection administered to rabbits in the preparationof antibodies.

Table 2

Titre of affinity purified anti-cpn10 peptide antibodies and controlanti-ovalbumin antibodies.

Table 3

Effect of passive immunization of confirmed-mated mice at days 1 and 2p.c., with antibodies to cpn10-derived peptides, on the number ofimplanted embryos and corpora lutea present at day 7 p.c.

* (Heteroscedastic t-test).

FIGURE LEGENDS

FIG. 1a

Purification of EPF. Heat extracted human platelets (100 units) werefractionated on SP-Sephadex and Heparin Sepharose, then applied to aTSK-Phenyl 5PW column and eluted with a reverse salt gradient. Fractionswere tested in the rosette inhibition test (based on EPF's capacity toaugment the rosette inhibiting activity of an immunosuppressiveantilymphocyte serum).

FIG. 1b

Active fractions (∩) from (a) were fractionated by PR-HPLC-1.

FIG. 1c

Active fractions (∩) from (b) were fractionated by RP-HPLC-2.

FIG. 1d

Active fractions (∩) from (c) were fractionated by RP-HPLC-3.

FIG. 1e

Interaction of immobilised monoclonal anti-EPF antibody 5/341 withactive fractions from (d) and equivalent fractions from human pregnancyserum, 6 d gestation (10 ml); human pregnancy urine, up to 1 monthgestation (10 liter); medium condition by oestrous mouse ovaries (100 )stimulated with prolactin and mouse embryo-conditioned medium (ovaryCM); serum free medium conditioned by the bovine kidney cell line MDBK(MDBK-CM; ATCC CCL 22, 10 liter); rat serum obtained 24 h post-partialhepatectomy (post-pH, 10 ml); rat liver obtained 24 h post-pH (40 g);all fractionated as in (a) to (d). Anti-EPF bound and unbound fractionswere tested in the rosette inhibition test, specificity was demonstratedby comparison with a parallel experiment using irrelevant antibody inwhich activity was not bound.

FIG. 2a

Analysis of EPF purified from 300 units human platelets as in FIG. 1A.Determination of monomeric size. Iodinated EPF was fractionated bySDS-PAGE,²⁹ the gel sliced (2 mm wide slices) and the distribution ofradioactivity and biological activity compared. (Inset) Direct CoomassieBlue staining of the same preparation.

FIG. 2b

Ion-spray mass spectrum of EPF, displayed as multiply protonatedmolecular ions. (Inset) Computer reconstruction as molecular mass.

FIG. 2c

Amino acid sequence (single letter code) of peptides derived from humanEPF, compared with rat cpn10 (underlined). EPF was digested withendoproteinase lys C and endoproteinase glu C, the resultant peptidessepareted by RP-HPLC and sequenced. The sequence of individual fragmentsis shown; all except 74-101 were derived from the lys digest (SEQ IDNO:22-25 are shown in this figure).

FIG. 3

Interaction of EPF and cpn60 (groEL).

FIG. 3a

Peak fractions in the excluded volume of a TSK G3000SW gel permeationcolumn, following application of a cpn60-EPF mixture +Mg²⁺ ATP, wereanalysed by SDS-PAGE (Schagger et al., 1987) and stained with silver(Morrissey, 1981). Left lane, +ATP; right lange -ATP. (Cpn60 is adecatetramer, M. 840 000; column exclusion limit >300 000. Higher M_(r)bands on SDS gel are oligomeric forms of groEL).

FIG. 3b

Immobilised cpn60 was mixed with human pregnancy serum (6 d gestation)in the presence or absence of Mg²⁺ ATP. Unbound and bound fractions (thelatter recovered from the gel by removal of ATP with EDTA) were thentested in the rosette inhibition test. Results are expressed as limitingdose, the highest dilution of sample giving a positive result in therosette inhibition test.

FIG. 4a, FIG. 4b, FIG. 4c

Rabbit antibodies to cpn10-peptide/ovalbumin conjugates. Antibodiestested in an ELISA against immunising antigens.

FIG. 5

Anti-N-peptide, anti-EPF #816, anti-EPF #810 and control anti-ovalbuminantibodies (100 ng/ml) were tested in an ELISA against □ N-peptide (5μg/ml) and □ EPF/cpn10 (1 μg/ml). Bound IgG was detected by thebiotin-streptavidin system (Amersham) with o-phenylene diamine assubstrate. Absorbance was read at 492 nm.

FIG. 6a

pRM1

FIG. 6b

pRM2

FIG. 6c

pRM3

FIG. 7

Preparation of antibodies to cpn10. Fusion protein (GST:rcpn10).

FIG. 8

Detection of anti-cpn10 antibodies in rabbit serum by ELISA. Serumharvested after the 4th booster dose of antigen.

FIG. 9

Relative [³ H]thymidine uptake (--▪--) and viability (--□--) of B16melanoma cells after incubation for 96 h with anti-cpn10-derived peptideantibodies. Proliferation was assessed by uptake of [³ H]thymidine intocells incubated with antibody, expressed as a percentage of [³H]thymidine incubated without antibody.

*p<0.05, **p<0.01, ***p<0.001, (Student's t test) n=3.

FIG. 10

Relative [³ H]thymidine uptake (--▪--) and viability (--□--) of MCA-2fibrosarcoma cells after incubation for 96 h with anti-cpn10-derivedpeptide antibodies. Proliferation was assessed by uptake of [³H]thymidine into cells incubated with antibody, expressed as apercentage of [³ H]thymidine incubated without antibody.

*p<0.05, **p<0.01, ***p<0.001, (Student's t test) n=3.

FIG. 11

Anti-cpn10 I-peptide Abs detect cpn10 on the surface of human Molt 4leukaemia cells.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 26                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Ala Gly Gln Ala Phe Arg Lys Phe Leu Pro Le - #u                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/product= "Other"R INFORMATION:                                              #"The Xaa at position 1 is acetyl."                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Xaa Ala Gly Gln Ala Phe Arg Lys Phe Leu Pr - #o Leu                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Glu Lys Ser Gln Gly Lys Val Leu Gln Ala Th - #r                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Xaa Ala Gly Gln Ala Phe Arg Lys Phe Leu Pr - #o Leu Ala                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Ala Gly Gln Ala Phe Arg Lys Phe Leu Pro Le - #u Ala                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Xaa Ala Gly Gln Ala Phe Arg Lys Phe Leu Pr - #o Leu                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/product= "Other"R INFORMATION:                                              #"The Xaa at position 1 is acetyl."                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Xaa Xaa Ala Gly Gln Ala Phe Arg Lys Phe Le - #u Pro Leu Xaa                 #                10                                                           - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/product= "Other"R INFORMATION:                                              #"The Xaa at position 1 is acetyl."                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Xaa Ala Gly Gln Ala Phe Arg Lys Phe Leu Pr - #o Leu Xaa                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/product= "Other"R INFORMATION:                                              #"The Xaa at position 1 is acetyl."                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Xaa Xaa Ala Gly Gln Ala Phe Arg Lys Phe Le - #u Pro Leu                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Xaa Glu Lys Ser Gln Gly Lys Val Leu Gln Al - #a Thr Xaa                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Glu Lys Ser Gln Gly Lys Val Leu Gln Ala Th - #r Xaa                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Xaa Glu Lys Ser Gln Gly Lys Val Leu Gln Al - #a Thr                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/product= "Other"R INFORMATION:                                              #"The Xaa at position 1 is acetyl."                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Xaa Ala Gly Gln Ala Phe Arg Lys Phe Leu Pr - #o Leu Cys                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Glu Lys Ser Gln Gly Lys Val Leu Gln Ala Th - #r Cys                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Val Leu Asp Asp Lys Asp Tyr Phe Leu                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                # 20               CRTC                                                       - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #   17             C                                                          - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #   17             T                                                          - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #           30     GGAC AAGCGTTTAG                                            - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #              26  CGTA CTTTCC                                                - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 104 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Gly Ser Met Ala Gly Gln Ala Phe Arg Lys Ph - #e Leu Pro Leu Phe Asp         #                15                                                           - Arg Val Leu Val Glu Arg Ser Ala Ala Glu Th - #r Val Thr Lys Gly Gly         #            30                                                               - Ile Met Leu Pro Glu Lys Ser Gln Gly Lys Va - #l Leu Gln Ala Thr Val         #        45                                                                   - Val Ala Val Gly Ser Gly Ser Lys Gly Lys Gl - #y Gly Glu Ile Gln Pro         #    60                                                                       - Val Ser Val Lys Val Gly Asp Lys Val Leu Le - #u Pro Glu Tyr Gly Gly         #80                                                                           - Thr Lys Val Val Leu Asp Asp Lys Asp Tyr Ph - #e Leu Phe Arg Asp Gly         #                95                                                           - Asp Ile Leu Gly Lys Tyr Val Asp                                                         100                                                               - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 43 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - Lys Val Leu Xaa Ala Thr Val Val Ala Val Gl - #y Ser Gly Ser Lys Glu         #                15                                                           - Tyr Gly Gly Thr Lys Val Val Xaa Xaa Xaa Xa - #a Asp Xaa Phe Leu Phe         #            30                                                               - Arg Asp Gly Asp Ile Leu Gly Lys Tyr Val As - #p                             #        40                                                                   - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 28 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - Lys Ser Gln Gly Lys Val Leu Gln Ala Thr Va - #l Val Ala Val Gly Xaa         #                15                                                           - Gly Xaa Lys Val Leu Leu Pro Glu Tyr Gly Gl - #y Thr                         #            25                                                               - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 47 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - Lys Phe Leu Pro Leu Phe Asp Arg Val Leu Va - #l Glu Lys Gly Gly Ile         #                15                                                           - Met Leu Pro Glu Lys Xaa Gln Gly Lys Val Va - #l Leu Asp Asp Lys Asp         #            30                                                               - Tyr Phe Leu Phe Arg Asp Gly Asp Ile Leu Gl - #y Lys Tyr Val Asp             #        45                                                                   - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 102 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/product= "Other"R INFORMATION:                                              #"The Xaa at position 1 is acetyl."                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                - Xaa Ala Gln Ala Gly Phe Arg Lys Phe Leu Pr - #o Leu Phe Asp Arg Val         #                15                                                           - Leu Val Glu Arg Ser Ala Ala Glu Thr Val Th - #r Lys Gly Gly Ile Met         #            30                                                               - Pro Leu Glu Lys Ser Gln Gly Lys Val Leu Gl - #n Ala Thr Val Val Ala         #        45                                                                   - Val Gly Ser Gly Gly Lys Gly Lys Gly Gly Gl - #u Ile Gln Pro Val Xaa         #    60                                                                       - Xaa Lys Xaa Gly Xaa Xaa Val Leu Leu Pro Gl - #u Tyr Gly Gly Thr Lys         #80                                                                           - Val Val Leu Asp Asp Lys Asp Tyr Phe Leu Ph - #e Arg Asp Gly Asp Ile         #                95                                                           - Leu Gly Lys Tyr Val Asp                                                                 100                                                               - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - Ala Gly Gln Ala Phe Arg Lys Phe Leu Pro Le - #u Cys                         #                10                                                           __________________________________________________________________________

What is claimed is:
 1. A method for suppressing cellular growthincluding the step of administration of an antibody selected from thegroup consisting of:(a) an antibody raised against recombinant cpn10which has the amino acid sequence GSMAGQAFRKFLPLFDRVLVERSAAETVTKGGIMLPEKSQGKVLQATVVAVGSGSKGKGGEIQPVSVKVGDKVLLP EYGGTKVVLDDKDYFLFRDGDILGKYVD (SEQID NO:21); (b) an antibody raised against a peptide Ac-AGQAFRKFLPLC (SEQID NO:13); AGQAFRKFLPLC (SEQ ID NO:26); EKSQGKVLQATC SEQ ID NO:14); and(c) an antibody raised against the following peptides(i) AGQAFRKFLPL(SEQ ID NO:1) (ii) Ac-AGQAFRKFLPI (SEQ ID NO:2) (iii) EKSQGKVLQAT (SEQID NO:3)and any one of peptides i, ii and iii modified by a single aminoacid deletion, addition or substitution, wherein the antibody is capableof suppressing cellular growth.
 2. A method as claimed in claim 1 forsupprssion of growth of tumour cells.
 3. A method as claimed in claim 2wherein the cells are leukaemia cells.
 4. A method according to claim 1wherein said antibody is selected from the group consisting:(a) anantibody raised against recombinant cpn10 which has the amino acidsequence GSMAGQAFRKFLPLFDRVLVERSAAETVTKGGIMLPEKSQGKVLQATVVAVGSGSKGKGGEIQPVSVKVGDKVLLP EYGGTKVVLDDKDYFLFRDGDILGKYVD (SEQID NO:21); (b) an antibody raised against a peptide Ac-AGQAFRKFLPLC (SEQID NO:13); AGQAFRKFLPLC (SEQ ID NO:26); EKSQGKVLQATC (SEQ ID NO:14); and(c) an antibody raised against the following peptides:(i) AGQAFRKFLPL(SEQ ID NO:1) (ii) Ac-AGQAFRKFLPL (SEQ ID NO:2) (iii) EKSQGKVLQAT (SEQID NO:3).
 5. A method as claimed in claim 1, which is a method ofterminating pregnancy.